Process for the isolation and purification of isoflavones

ABSTRACT

The present invention relates to a process for the isolation and purification of isoflavones from a number of different biomass sources. More particularly, the present invention relates to a three-step process whereby a biomass containing isoflavones with a solvent thereby forming an extract that is subsequently fractionated using a reverse phase matrix in combination with a step gradient elution, wherein the resulting fractions eluted from the column contain specific isoflavones that are later crystallized. The purified isoflavone glycosides may then be hydrolyzed to their respective aglycone.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for the isolation andpurification of isoflavones from a number of different biomass sources.More particularly, the present invention relates to a three-step processwhereby specific isoflavones are isolated and purified from plantmaterials such as Glycine max (soy) and Pueraria lobata (kudzu).

2. Description of the State of Art

The isoflavones are a group of naturally occurring plant compoundshaving the aromatic heterocyclic skeleton of flavan and which oftenpossess estrogenic activity. Their existence in Glycine, Pueraria andother plant species has long been known. The roots of Pueraria are usedunder the name of Gegen in Chinese traditional medicine as the maincomponent of a prescription, Gegen Tao, for the common cold. ThePueraria root contains several isoflavone compounds, such as daidzin(1a), puerarin (1f), and daidzein (2a), shown in FIGS. 1 and 2,respectively. Soy is known to contain the isoflavones, daidzin (1a),genistin (1b), glycitein (1c), 6"dadidzin-O-acetyl (1d), 6"-O-acetylgenistin (e), 6"-O-malonyl daidzin (1g), 6"-O-malonyl genistin (1h),also shown in FIG. 1. It has been reported that these isoflavonecompounds possess antihemolytic, antioxidative, antifungal, estrogenicand antitumor activities, and also are the source of the undesirablebitter and astringent tastes characteristic in soybean. Recent studiessuggest that the isoflavones daidzin and daidzein work to suppress thedesire for alcohol; while genistin and genistein may block the formationof new blood vessels which are needed to nourish malignant tumors.

A variety of different procedures for isolating and purifyingisoflavones from plant materials have been published. The reference thatis most prevalently cited throughout the literature is Ohshima et al.,Planta Medica, 250-254 (1988). Ohshima at al., discloses a method ofextracting Pueraria root, using acetone to first extract thenon-glycosidic flavonoids. The dried root is then extracted withmethanol, the methanolic extract is subsequently re-extracted withaqueous butanol, and the resulting butanol layer is passed over achromatographic column, eluting with methanol. The resulting glycosidicfractions are further chromatographed over a silica gel column followedby two preparative high performance liquid chromatography columns. Ohtaet al., Agric Biol. Chem., 43:7, 1415-1419 (1979) discloses a method ofisolating and purifying isoflavones from defatted soybeans whereby thedefatted soybeans are extracted with ethanol and the resulting ethanolextracts are treated with acetone and ethyl acetate. The ethyl acetateextract is then fractionated over silica gel and Sephadex LH-20 columnsfollowed by multiple recrystallizations. Farmakalidis et al., reportedin the Journal of Agric. Food Chem., 33:385-389 (1985) that acetone whenmixed with 0.1N HCl was superior over 80% methanol as an extractionsolvent. The subsequent isolation procedure followed by Farmakalidis etal. was that of Ohta et al., discussed previously. E. D. Walterpublished a method for the extraction and isolation of genistin and itsaglycone, genistein, from soybeans, J. Amer. Chem. Soc., 63:3273-3276(1941). 10 Kg of defatted soybean flakes having been extracted withhexane are twice extracted using methanol. Acetone is added to thecombined methanolic extract to precipitate some of the phosphatides andother impurities. The supernatant is decanted and two volumes of waterare added to precipitate out the genistin. Multiple recrystallizationsare then performed to purify the genistin. The above technical papers byOhshima et al., Ohta et al., and Farmakalidis et al. are just a fewexamples of the many processes that currently exist in the literature,whereby isoflavones are extracted, isolated and purified from variousplant materials. However each process disclosed involves multiple stepsand various solvents. Consequently, the disclosed laboratory scaleprocesses are not easily scaled up to an efficient commercial processwhere disposal considerations of various solvents play an important rolein the overall feasibility of the process. A further disadvantage of theprocesses as disclosed in the literature is the requirement of multiplechromatography columns. The eluants utilized by various researchers inthe field typically separate the isoflavones from other compoundspresent in the plant extract. However, upon isolation of the isoflavonecompounds further separation techniques involving chromatography arerequired. These separation techniques necessitate the continuous need tomonitor the eluant as it runs off the column, thus making it possible tocollect those fractions of eluant that contain a particular isoflavone.The Walter article which eliminated the necessity of chromatography onlyisolated one isoflavone, genistin.

There is still a need, therefore, for a process and procedure forisolating and purifying isoflavones from isoflavone containing biomassin a commercially viable manner which directly provides a highconcentration of the various isoflavones which can be subsequentlyrecovered in high yield and purity.

SUMMARY OF THE INVENTION

Accordingly, it is an object of this invention to provide a simplifiedmethod for the extraction, isolation and purification of specificisoflavones.

Another object of the present invention is to convert resultingisoflavone glycosides to their respective aglycones.

Additional objects, advantages, and novel features of this inventionshall be set forth in part in the description and examples that follow,and in part will become apparent to those skilled in the art uponexamination of the following specification or may be learned by thepractice of the invention. The objects and advantages of the inventionmay be realized and attained by means of instrumentalities,combinations, compositions, and methods particularly pointed out in theappended claims.

To achieve the foregoing and other objects and in accordance with thepurposes of the present invention, as embodied and broadly describedtherein the method of this invention comprises contacting any plantmaterial containing isoflavones with a solvent thereby forming anextract that is subsequently fractionated using a reverse phase matrix,wherein the resulting fractions eluted from the column contain specificisoflavones that are later crystallized. The purified isoflavoneglycosides may then be hydrolyzed if so desired to their respectiveaglycones.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specifications, illustrate the preferred embodiments of the presentinvention, and together with the description serve to explain theprinciples of the invention.

In the Drawing:

FIG. 1 shows the structure of specific isoflavones.

FIG. 2 shows the resulting structure, daidzein, upon hydrolysis ofdaidzin.

FIG. 3 shows the resulting structure, genistein, upon hydrolysis ofgenistin.

FIG. 4 shows a chromatographic separation of daidzin and genistin fromdefatted soy flakes.

FIG. 5 shows a flow diagram for the extraction and isolation procedurefor isoflavones from soy products.

FIG. 6 shows a flow diagram for the extraction and isolation procedurefor isoflavones from Pueraria.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In general the present invention relates to a high yield process for theisolation and purification of the isoflavones appropriate to aparticular plant material which includes but is not limited to partiallypurified plant derivatives, shown in FIGS. 1, 2 and 3. The purifiedisoflavone glycosides may then be easily converted by acid hydrolysis totheir respective aglycone. The preferred embodiment of the presentinvention is a three step process and is described in detail below. Thefirst step includes contacting a plant material that containsisoflavones with a solvent, thus resulting in a crude extract containinga mixture of compounds that includes isoflavones. The second stepinvolves adsorbing the isoflavone compounds onto a reverse phase matrixfollowed by the specific desorption of certain isoflavones from thematrix in fractions using a step gradient elution, and then evaporatingselected pools. The third and final step involves crystallization toachieve a final high purity product. The thus purified isoflavoneglycosides may then, if desired, be contacted with an acid toeffectively cleave the glucose molecule, resulting in an aglycone thatcan be subsequently crystallized.

This invention includes a process for the extraction, isolation andpurification of isoflavones from plant materials or biomasses thatcontain isoflavones. As way of illustration only, the following is apartial list of plant sources containing isoflavones: Pueraria lobata,Glycine max, Thermospin sp., Baptisia sp., Trifolium sp., etc. Again,this list is exemplary of the plant materials that contain isoflavones,and is not meant to limit the scope of plant materials which may beutilized by the present invention. The first step in the process of thepresent invention, extraction of the isoflavone compounds, is preferablyaccomplished by mixing or contacting a first solvent, such as analcohol, and preferably ethanol or methanol with a plant materialcontaining isoflavones. Depending on the type of plant material used, itmay be necessary to grind it into a range of 0.1-10 min. The degree ofcomminutation of the plant material should provide sufficientparticulate surface area for the first solvent to contact, but againthis depends on the type of plant material used. The skilled person inthis art will recognize that a variety of extraction methods areavailable in the literature, such as, percolation, vat extraction,counter-current extraction, etc. The particular method of extractionemployed is not essential to the process of the present invention. Inthe extraction process, the temperature of extraction is between 40°-70°C., with 50°-60° C. being preferred. The mount of plant material tosolvent mixture used in the extraction process varies between 1:1 to1:10 on a gram:milliliter basis, with 1:1 to 1:3 being preferred. Theisoflavones and some of the extraneous materials that are contained inthe comminuted plant material are soluble in the first solvent used.Thus, the first solvent, the isoflavones and some of the extraneousmaterials form the crude extract. The crude alcohol extract is nextdiluted with distilled water to a final volume of 20% alcohol in water.The resulting solution is then filtered to remove insoluble materials.

After completion of the formation of the crude extract, the second step,the separation of the isoflavones, begins. Since the crude extractcontains not only the desired isoflavones but also extraneous materialsthat are soluble in the first solvent of the crude extract, it isdesirable to recover the isoflavones with as little extraneous materialas possible. The following step, of the isolation of the isoflavonesfrom the crude extract, includes the partial elimination of unwantedextraneous materials such as phenols, H₂ O, proteins, simple and complexsugars, etc., while selectively maintaining the isoflavones. To recoverthe isoflavones the water diluted crude extract is loaded onto a reversephase matrix. In this step the preferred adsorbent is polymethacrylateor C-18. Subsequent to adsorbing the isoflavones to the matrix, groupsof isoflavones are specifically desorbed from the matrix and eluted infractions using a series of specific solvents each having a volume fivetimes that of the column that supports the matrix and comprising aspecific ratio of a second solvent, such as an alcohol to water. Thepreferred alcohol is methanol or ethanol. Molecules having the lowestaffinity for the adsorbent are specifically desorbed passing directlythrough the matrix and collected in the first wash column volume. Witheach successive column volume of eluting solvent the specific ratio ofthe second solvent to water increases and the isoflavones arespecifically desorbed in sequence according to their affinity for theadsorbent. FIG. 4 illustrates a typical chromatogram for soy havingretention values in column volumes for the various isoflavones. Thehorizontal axis represents the total volume of eluting solvent putthrough the column. Each step in the gradient has a volume five timesthat of the column; therefore, 0-5.0 column volumes represents the firststep in the gradient, while 5.1-10 column volumes represents the secondstep in the gradient and so on. As shown in FIG. 4, the first major peakbegins at approximately column volume 3.8 and ends around column volume5.0. The isoflavone represented by that peak is said to elute in a totalof 1.2 column volumes. Since these numbers fall between 0 and 5.0 it canalso be determined which step in the gradient and thus the ratio of thesecond solvent to water necessary to desorb that specific isoflavone. Itis important that the ratio of the second solvent to water increaseswith each step as opposed to decreasing. If a large alcohol ratio wasused in the first step, those isoflavones having a higher relativeaffinity toward the adsorbent would desorb along with those isoflavoneshaving a lower relative affinity for the adsorbent and thus noseparation would be achieved. Each pool collected represents a distinctmajor isoflavone. The individual pools are evaporated resulting in asolid or crude product of each desired isoflavone.

The preferred third and final step in the process is the crystallizationor final purification of each isoflavone. To begin, the dried crudeproduct of one isoflavone from the preceding step is dissolved (1 mg/ml)in a volume of solvent, the preferred solvent is methanol, howeverethanol will also suffice. The resulting suspension is decolorized bythe addition of active carbon that is subsequently removed by passingthe suspension through a filter aid, such as celite. The volume of thedecolorized solution is evaporated to a final ratio of 10-60 mg ofisoflavone per ml of solvent and is then crystallized by refrigeratingthe solution overnight. The refrigerated suspension is then passedthrough a filter and the final product, the crystallized isoflavone, iscollected and dried.

There is some evidence in the research literature that the isoflavoneaglycones may have more biological activity than their respectiveglycosides. However, isoflavone aglycones are found in very lowabundance in plant tissues. Therefore, the present invention alsocontemplates the hydrolysis of the isoflavone glycosides to form theirrespective aglycones. Cleavage of the glucose, acetylglucose ormalonylglucose molecule from an isoflavone glycoside may be accomplishedby subjecting the isoflavone glycoside to acid hydrolysis in HCl-water,preferably 4N HCl at 100° C. for 5 hours. The benefits of an acid/waterhydrolysis are the elimination of organic solvents and ease of recoveryof the product by filtration. Following the hydrolysis, the isoflavoneaglycone is recovered by filtration, dried, and may then be crystallizedfrom methanol, as discussed above, or alternatively extracted withdiethyl ether, evaporated, and recrystallized from alcohol as discussedin further detail in the Examples which follow.

The following non-limited examples provide specific high yield processesfor isolating and purifying isoflavones from plant tissues. Allscientific and technical terms have the meanings as understood by onewith ordinary skill in the art. Melting points were measured on aMEL-TEMP II apparatus equipped with a Barnant 100 ThermocoupleThermometer and are uncorrected. HPLC was performed on a Hitachichromatograph (L-6200 Intelligent Pump, D-6000 Interface, L-4000 UVDetector and AS-4000 Intelligent Auto Sampler). Combinations ofacetonitrile and water (2% acetic acid) in different concentrations wereused as the HPLC solvent system. Commercially available chemicals wereused without any further purification. NMR was performed on a JEOLEclipse 400. Centrifugation was done on a Beckman Model CS 6KRcentrifuge.

EXAMPLE I

The entire isolation and purification procedure for isoflavones from soymolasses discussed in detail below is represented in the flow diagramdepicted in FIG. 5.

A. Methanol extraction of soy molasses:

Approximately 1544 grams of soy molasses were introduced into a beakerhaving 900 ml of methanol and a magnetic stir bar. The mixture was thenstirred at 50°-60° C. for 30 minutes and the solids were removed bycentrifugation for 30 minutes. These solids were reextracted with 900 mlof methanol at 50°-60° C. for 30 minutes, centrifuged, and the twosupernatants combined to form a crude extract. HPLC analysis of theextracts showed daidzin at 5.83 mg/ml with a purity of 1.3% and genistinat 6.46 mg/ml with a purity of 1.5%.

B. Specific adsorption/desorption separation using a reverse phasematrix:

About 1.8 L of the methanol extract of soy molasses was diluted withdistilled water to make a 20% methanol in water solution which wasloaded onto a properly conditioned 4"×70" polymethacrylate column andgradually eluted stepwise with five column volumes each of 50%, 60%, and75% methanol in water. The eluant was collected in 100 ml fractions asit came off the bottom of the column and analyzed for the presence ofisoflavones by HPLC. The first step gradient of 50% methanol in waterwas represented by a pool eluted between 0-5 total column volumes.Daidzin eluted from the column in the fraction which was represented bya pool eluted between 2.8-4.1 total column volumes. Genistin was elutedwith the second step gradient of 60% methanol in water in a pool elutedbetween 6.3-8.0 total column volumes. Glycitein was eluted from thecolumn using the third step gradient of 75 % methanol in water in a pooleluted between 11.9-13.3 total column volumes. The individual productpools were taken to dryness resulting in dry crude products.

C. Crystallization:

About 5.87 grams of daidzin crude product (62% purity) were dissolved in300 ml methanol, followed by the addition of active carbon to decolorizethe solution. The active carbon was removed by filtration and thesolution was evaporated to 120 ml and refrigerated overnight. Thesuspension was then filtered and the final product, 2.9 grams of daidzincrystals were recovered resulting in an 80% recovery having a 97%purity. Daidzin m.p. 225°-225.8° C. ¹ H NMR (400 MHz, DMSO-d₆) δ:6.83(2H, d, J=8 Hz, H-3'), 7.13 (1H, dd, J=8, 2 Hz, H-6), 7.23 (1H, d, J=2Hz, H-8), 7.42 (2H, d, J=8 Hz, H-2'), 8.06 (1H, d, J=8 Hz, H-5), 8.38(1H, s, H-2), 9.55 (1H, s, 4'-OH). ¹³ C NMR (100 MHz, DMSO-d₆) δ:153.8,124.2, 175.2, 127.5, 115.5, 161.4, 103.9, 157.5, 119.0, 122.8, 130.6,115.5, 157.8, 103.9, 73.6, 77.0, 70.1, 77.7, 60.9.

D. Hydrolysis and Crystallization:

987.3 mg of daidzin were refluxed in 470 ml 4N HCl for five hours at105°±5° C. About 554.1 mg of daidzein was recovered by filtration whichresulted in a 92% recovery. About 442.9 mg of this recovered daidzeinfrom the hydrolysis step was dissolved in 200 ml methanol andcrystallized in 30 ml methanol. About 339.0 mg daidzein crystal wasobtained with 77% recovery and about 99% purity.

EXAMPLE II

The entire isolation and purification procedure from defatted soy flakesdiscussed in detail below is represented in the flow diagram depicted inFIG. 5.

A. Methanol extraction of defatted soy flakes:

Approximately 909 grams of defatted soy flakes were introduced into abeaker having 2 L of methanol and a magnetic stir bar. The mixture wasthen stirred at 55° C. for 30 minutes and the solids were removed byfiltration. These solids were extracted four more times each with 2 L ofmethanol at 55° for 30 minutes, filtered and the five filtrates combinedto form the crude extract. HPLC analysis of the extract showed about 485mg daidzin and 636 mg genistin. Two earlier eluting peaks wereidentified as 6"-O-malonyl daidzin and 6"-O-malonyl genistin.

B. Specific adsorption/desorption separation using a reverse phasematrix:

About 9 L of defatted soy flakes methanol extract were diluted withdistilled water to make a 20% methanol in water solution which was thenloaded onto a properly conditioned 4"×70" polymethacrylate column andgradually eluted stepwise with five column volumes each of 50%, 60%, and75% methanol in water. The eluant was collected in 100 ml fractions asit came off the bottom of the column and analyzed for the presence ofisoflavones by HPLC. The first step gradient of 50% methanol in waterrepresented column volumes from 0-5. 6"-O-malonyl daidzin, 6"-O-malonylgenistin and daidzin eluted from the column in the fractions which wererepresented by a pool eluted between 0-1.3, 1.0-1.6 and 3.5-6.1 columnvolumes respectively. Genistin was eluted with the second step gradientof 60% methanol in water in a pool between 7.0-10.0 total columnvolumes. The individual pools were taken to dryness resulting in 396 mgof daidzin and 515 mg of genistin.

C. Crystallization:

About 0.727 grams of genistin crude product (63% purity) were dissolvedin 250 ml methanol, followed by the addition of active carbon todecolorize the solution. The active carbon was then removed byfiltration and the solution was evaporated to 22 ml and refrigeratedovernight. The suspension was then filtered and the final product, 0.339grams crystalline genistin were recovered with a 97% purity and 72%recovery. Genistin m.p.250.8°-252.0° C. ¹ H NMR (400 MHz, DMSO-d₆)δ:6.47 (1H, d, J=2 Hz, H-6), 6.72 (1H, d, J=2 Hz, H-8), 12.92 (1H, s,H-5), 6.83 (2H, d, J=8 Hz, H-3'), 7.39 (2H, d, J=8 Hz, H-2¹), 8.39 (1H,s, H-2), 9.61 (1H, s, 4¹ -OH), 12.92 (1H, s, 5-OH). ¹³ C NMR (100 MHz,DMSO-d₆) 154.6, 123.1, 181.0, 162.1, 100.1, 163.5, 95.0, 157.7, 106.6,121.5, 130.7, 115.6, 157.7, 100.4, 73.6, 76.9, 70.1, 77.7, 60.0.

D. Genistein via Hydrolysis and Crystallization:

About 49.0 mg of genistin were refluxed in 15 ml 4N HCl for five hoursat 105°±5° C. About 22.9 mg of genistein was obtained with about 75%recovery. About 13 mg genistein from the previous step was extractedtwice with 50 ml diethyl ether. The extracts were combined, andevaporated to dryness. The solids obtained were crystallized from 2 mlof a solution made from 3 parts ethanol and 2 parts water. About 8.2 mgof the genistein crystal was obtained with 63% recovery and about 99%purity.

EXAMPLE III

The genistin used in the present Example was obtained in the same manneras described in Example I above from soy molasses.

D. Hydrolysis and Crystallization:

About 1578 mg of genistin were refluxed in 960 ml 4N HCl for five hoursat 100°±5° C. About 948 mg genistein were obtained with about 96%recovery. 483.2 mg of genistein (99% purity) recovered from thehydrolysis step was dissolved in 450 ml methanol and crystallized in 20ml methanol. About 373.8 mg of genistein crystal was obtained giving a77% recovery and 99.5% purity. Genistein m.p. 298.4°-299.9° C. ¹ H NMR(400 MHz, DMSO-d₆) 6.22 (1H, d, J=2 Hz, H-8), 6.38 (1H, d, J=2 Hz, H-6),6.83 (2H, d, J=8 Hz, H-3'), 7.39 (2H, d, J=8 Hz, H-2'), 8.43 (1H, s,H-2), 9.61 (1H, s, 4'-OH), 10.90 (1H, s, 7-OH) 12.92 (1H, s, 5-OH). ¹³ CNMR (100 MHz, DMSO-d₆) 154.5, 122.8, 180.7, 162.5, 99.5, 164.8, 94.2,157.9, 105.0, 121.7, 130.7, 115.6, 158.1.

EXAMPLE IV

The entire isolation and purification procedure from dry kudzu rootdiscussed in detail below is represented in the flow diagram depicted inFIG. 6.

A. Ethanol extraction of dry kudzu Pueraria lobata root:

Approximately 137 grams of dry kudzu root, 1.68% (w/w) isoflavones indry biomass were introduced into a beaker having 600 ml of 95% ethanoland a magnetic stir bar. The mixture was then stirred at 55° C. for 30minutes and the solids were removed by centrifugation for 30 minutes.These solids were extracted two more times with 600 ml of methanol at55° C. for 30 minutes, centrifuged, and the three supernatants combinedand evaporated to form a erode extract. HPLC analysis of thesupernatants showed the total isoflavones in the extract to be 26%(w/w).

B. Specie adsorption/desorption separation using a reverse phase matrix:

About 50 ml of kudzu ethanol extract were diluted with distilled waterto make a 20% ethanol in water solution which was then loaded onto aproperly conditioned 1.5×20.3 cm polymethacrylate column and graduallyeluted stepwise with five column volumes each of 25%, 50%, 75%, and 85%methanol in water. The eluant was collected in 20 ml fractions as itcame off the bottom of the column and analyzed for the presence ofisoflavones by HPLC. The first step gradient of 25% methanol in waterwas represented by a pool eluting between 0-5 column volumes. The secondstep gradient of 50% methanol in water was represented by a pool elutingbetween 5-10 column volumes, etc. Puerarin and daidzin eluted from thecolumn in the fractions eluting between 6.7-9.0 and 9.2-10.9 totalcolumn volumes, respectively. Genistin was eluted with the third stepgradient of 75% methanol in water eluting in a pool between 12.2-13.8total column volumes. Daidzein and genistein were eluted from the columnusing the fourth step gradient of 85% methanol in water and wasrepresented by a pool eluting between 15.2-16.8, and 17.7-19.3 columnvolumes, respectively. Ethanol in water was also used as a gradientelution solvent in this case. The individual pools were taken to drynessresulting in a dry solid or crude product.

C. Crystallization:

The procedure set out in Example I above was followed.

D. Hydrolysis and Crystallization:

The procedure set out in Example I above was followed. Daidzein m.p.315.2-325.4 (dec.). ¹ H NMR (400 MHz, DMSO-d₆) 6.81 (2H, d, J=2 and 8Hz, H-3'), 6.86 (1H, d, J=2 Hz, H-8), 6.94 (1H, dd, J-=8, 2 Hz, H-6),7.43 (2H, d, J=8 Hz, H-2'), 7.97 (1H, d, J=8 Hz, H-5), 8.38 (1H, s,H-2), 9.5 (1H, s, 4'-OH), 10.7 (1H, s, 7-OH). ¹³ C NMR (100 MHz,DMSO-d₆) 153.3, 124.0, 175.2, 127.8, 115.4, 163.0, 102.6, 157.9, 117.1,123.0, 130.6, 115.6, 157.7.

The foregoing description is considered as illustrative only of theprinciples of the invention. Furthermore, since numerous modificationsand changes will readily occur to those skilled in the art, it is notdesired to limit the invention to the exact construction and processshown as described above. Accordingly, all suitable modifications andequivalents may be resorted to falling within the scope of the inventionas defined by the claims which follow.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method of isolatingisoflavones from any plant material that contains one or more type ofisoflavone compounds, comprising the steps of:contacting the plantmaterial for a selected period of time with a first solvent whereby atleast some of said isoflavone compounds are soluble and transported intosaid first solvent thereby forming a crude extract; passing said crudeextract through a column wherein said column contains an absorbent forspecifically adsorbing said isoflavone compounds; desorbing saidisoflavone compounds sequentially from said adsorbent by flowing aseries of eluant mixtures making up a step gradient elution over saidcolumn; and collecting each of the individual eluant mixtures of saidstep gradient elution flowing through said column wherein each of saidindividual eluant mixture contains primarily isoflavone compound.
 2. Themethod of claim 1, wherein said individual eluant mixtures have specificratios of a second solvent in the range of 25-85% to water.
 3. Themethod of claim 1, wherein said first solvent is an alcohol.
 4. Themethod of claim 2, wherein said second solvent is an alcohol.
 5. Themethod of claim 4, wherein said alcohol is methanol.
 6. The method ofclaim 4, wherein said alcohol is ethanol.
 7. The method of claim 1,wherein said adsorbent is a reverse phase matrix.
 8. The method of claim7, wherein said reverse phase matrix is polymethacrylate.
 9. The methodof claim 7, wherein said reverse phase matrix is C-18.
 10. The method ofclaim 1, wherein the plant material is soy.
 11. The method of claim 1,wherein said plant material is soy molasses.
 12. The method of claim 1,wherein said plant material is defatted soy flakes.
 13. The method ofclaim 1, wherein said plant material is kudzu root.
 14. The method ofclaim 1, further comprising the step of drying said individual eluantmixtures to obtain a crude product.
 15. The method of claim 14, furthercomprising the step of crystallizing said crude product.
 16. The methodof claim 15, further comprising the step of hydrolyzing said crystallineproduct.
 17. The method of claim 16 further comprising the step ofcrystallizing said hydrolyzed product.
 18. A method of isolatingisoflavones from any plant material that contains one or more isoflavonecompounds, comprising the steps of:contacting the plant material for aselected period of time with a first solvent whereby at least some ofsaid isoflavone compounds are soluble or transported into said firstsolvent thereby forming a crude extract; passing said crude extracthaving one or more of said isoflavone compounds through a low pressurecolumn wherein said low pressure column contains an adsorbent forspecifically adsorbing said isoflavone compounds; desorbing saidisoflavone compounds one at a time from said adsorbent by sequentiallyflowing a series of eluants over said adsorbent, wherein each eluant insaid series comprises a specific ratio of a second solvent to water thatspecifically desorbs and transports at least one isoflavone compound;and collecting separately, each of said eluants transporting a specificisoflavone compound.
 19. The method of claim 18, wherein each of saideluants are evaporated to a dry solid.
 20. The method of claim 18,wherein said first solvent is an alcohol.
 21. The method of claim 19,wherein said second solvent is an alcohol.
 22. The method of claim 21,wherein said alcohol is methanol.
 23. The method of claim 21, whereinsaid alcohol is ethanol.
 24. The method of claim 18, wherein saidadsorbent is a reverse phase matrix.
 25. The method of claim 24, whereinsaid reverse phase matrix is polymethacrylate.
 26. The method of claim24, wherein said reverse phase matrix is C-18.
 27. The method of claim18, wherein the plant material is a soy.
 28. The method of claim 18,wherein said plant material is soy molasses.
 29. The method of claim 18,wherein said plant material is defatted soy flakes.
 30. The method ofclaim 18, wherein said plant material is kudzu root.
 31. The method ofclaim 19, further comprising the step of crystallizing said dry solid.32. A method of isolating isoflavones from any plant material thatcontains one or more isoflavone compounds, comprising the stepsof:extracting one or more isoflavone compounds from the plant material;adsorbing said isoflavone compounds to a reverse phase matrix; desorbinga first isoflavone compound from said reverse phase matrix by flowing afirst eluant having a ratio of a solvent to water over said matrix;collecting said first eluant after said first eluant has flowed oversaid matrix; desorbing a second isoflavone compound from said reversephase matrix by flowing a second eluant, having a ratio of said solventto water which is higher than the ratio of said solvent to water makingup said first eluant, over said matrix; and collecting said secondeluant after said second eluant has flowed over said matrix.
 33. Themethod of claim 1, wherein said column is for low pressurechromatography.
 34. The method of claim 31, further comprising the stepof hydrolyzing said crystallized product.
 35. The method of claim 1,further comprising the step of diluting said crude extract with waterprior to passing said crude extract through said column.